Genome-wide profiling of non-smoking-related lung cancer cells reveals common RB1 rearrangements associated with histopathologic transformation in EGFR-mutant tumors.

BACKGROUND: The etiology and the molecular basis of lung adenocarcinomas (LuADs) in nonsmokers are currently unknown. Furthermore, the scarcity of available primary cultures continues to hamper our biological understanding of non-smoking-related lung adenocarcinomas (NSK-LuADs). PATIENTS AND METHODS: We established patient-derived cancer cell (PDC) cultures from metastatic NSK-LuADs, including two pairs of matched EGFR-mutant PDCs before and after resistance to tyrosine kinase inhibitors (TKIs), and then performed whole-exome and RNA sequencing to delineate their genomic architecture. For validation, we analyzed independent cohorts of primary LuADs. RESULTS: In addition to known non-smoker-associated alterations (e.g. RET, ALK, EGFR, and ERBB2), we discovered novel fusions and recurrently mutated genes, including ATF7IP, a regulator of gene expression, that was inactivated in 5% of primary LuAD cases. We also found germline mutations at dominant familiar-cancer genes, highlighting the importance of genetic predisposition in the origin of a subset of NSK-LuADs. Furthermore, there was an over-representation of inactivating alterations at RB1, mostly through complex intragenic rearrangements, in treatment-naive EGFR-mutant LuADs. Three EGFR-mutant and one EGFR-wild-type tumors acquired resistance to EGFR-TKIs and chemotherapy, respectively, and histology on re-biopsies revealed the development of small-cell lung cancer/squamous cell carcinoma (SCLC/LuSCC) transformation. These features were consistent with RB1 inactivation and acquired EGFR-T790M mutation or FGFR3-TACC3 fusion in EGFR-mutant tumors. CONCLUSIONS: We found recurrent alterations in LuADs that deserve further exploration. Our work also demonstrates that a subset of NSK-LuADs arises within cancer-predisposition syndromes. The preferential occurrence of RB1 inactivation, via complex rearrangements, found in EGFR-mutant tumors appears to favor SCLC/LuSCC transformation under growth-inhibition pressures. Thus RB1 inactivation may predict the risk of LuAD transformation to a more aggressive type of lung cancer, and may need to be considered as a part of the clinical management of NSK-LuADs patients.

Evaluation of the effects of feeding dried algae containing beta-1,3-glucan on broilers challenged with Eimeria.

This study determined the effect of a dried algae product containing beta-1,3-glucan on broiler performance and immunity during an Eimeria challenge. Heterotrophically grown Euglena gracilis, which contained ∼55% beta-1,3-glucan, was dried and milled for inclusion into a non-medicated starter diet. Two experiments were conducted to evaluate dietary treatments containing 0, 50, 100, 150, and 200 g/ton dried algae. In both experiments, male broilers were orally challenged on day 14 with a coccidial inoculum consisting of E. acervulina, E. maxima, and E. tenella. Fecal matter was collected 120-144 hours post-exposure to determine relative amounts of oocyte shedding and birds were sacrificed on day 20 for lesion scoring. Broiler performance was assessed on a weekly basis. In the first experiment, birds receiving dried algae at 50 and 200 g/ton showed a significant improvement in FCR compared to the infected control during the challenge period (days 14-20). In the second experiment, the dried algae treatment had no significant effect on FCR, but lesion scores were significantly reduced in the groups receiving 50, 150, and 200 g/ton dried algae relative to the infected control. In both experiments, the dried algae treatment did not significantly impact mortality or oocyte shedding. In the second experiment, staining of intestinal samples with fluorescently tagged antibodies demonstrated that dried algae at 100 g/ton increased the number of intestinal macrophages compared to the infected control. A significant and dose-dependent increase in intestinal MHC-II+ expression was also observed for birds fed dried algae, with an 8-fold increase observed in the 200 g/ton group relative to the infected control. Similarly, increased total immune cell density (measured by the mean fluorescence intensity of CD45+ cells) was also observed at 150 and 200 g/ton. Overall, these data suggest that dried algae rich in beta-1,3-glucan can help improve gut immunity and host protection, thereby reducing morbidity associated with coccidiosis.

Effects of tannic acid extract on performance and intestinal health of broiler chickens following coccidiosis vaccination and/or a mixed-species Eimeria challenge.

Two experiments were conducted to investigate the effects of tannic acid extract (TAE) formulations on the performance and intestinal health of male Cobb × Cobb 500 broilers exposed to coccidiosis. In the first experiment, 320 broiler chicks were randomly assigned to 5 treatments with 8 replicates. Treatments included non-medicated, uninfected (NC); non-medicated, infected (PC); salinomycin (SAL, 66 mg/kg); tannic acid (TA, 0.5 g/kg) and TAE (TAE, 0.5 g/kg). On d 14, all groups (except NC) were orally inoculated with Eimeria acervulina, E. maxima and E. tenella oocysts. Intestinal lesion scores, fecal oocyst counts (OPG) and performance were evaluated on d 20. The PC had greater lesions and higher FCR than infected, supplemented groups. Only TAE reduced OPG compared to PC (P < 0.05). In the second experiment, 3,000 broiler chicks were vaccinated on day of hatch with live coccidial oocysts, then randomly assigned to 5 treatments with 15 replicates. Treatments included non-medicated (CNT); salinomycin (SAL, 66 mg/kg); robenidine (ROB, 33 mg/kg); TAE (0.5 g/kg) and TAE with Bacillus coagulans (TAE+BC, 0.5 g/kg). On d 29, a subset of pens (n = 20) were challenged with a mixed Eimeria spp. oral inoculum; performance, lesions and OPG were evaluated on d 35. An immune challenge was created in half the pens by issuing broilers feed without supplementation materials during the challenge. For the non-challenged pens (n = 55), performance was measured up to d 49. Performance of non-challenged, vaccinated-CNT birds was improved with all treatments at d 21 and d 49. Among the challenged birds, withdrawal of SAL or ROB resulted in FCR similar to the challenged CNT group (P > 0.05), whereas withdrawal of TAE or TAE+BC maintained improved FCR compared to challenged-CNT birds (P < 0.05). These findings indicate supplementation of TAE and TAE+BC with coccidiosis vaccination can be considered as a potential alternative strategy to address coccidiosis in broiler chickens.

Platelets interact with tissue factor immobilized on surfaces: effects of shear rate.

BACKGROUND: While procoagulant activities of Tissue Factor (TF) have been widely investigated, its possible pro-adhesive properties towards platelets have not been studied in detail. MATERIAL AND METHODS: We explored the interaction of platelets with human Tissue Factor (hTF) firmly adsorbed on a synthetic surface of polyvinilidene difluoride (PVDF) using different shear rates. For studies at 250 and 600 s(-1), TF firmly adsorbed was exposed to flowing anticoagulated blood in flat perfusion devices. Deposition of platelets and fibrin were evaluated by morphometric, immunocytochemical and ultrastructural methods. Prothrombin fragment 1 + 2 (F1 + 2) levels were also measured. Experiments at 5000 s(-1), were performed on the Platelet Function Analyzer (PFA-100) with experimental cartridges with collagen (COL) or collagen-hTF (COL + TF). Haemostatic effect of recombinant activated FVIIa (rFVIIa) was assessed in the same experimental settings. RESULTS: Platelet deposition on hTF reached 19.8 +/- 1.3% and 26.1 +/- 3.4% of the total surface, at 250 and 600 s(-1), respectively. Fibrin formation was significantly higher at 250 s(-1) than at 600 s(-1) (P < 0.05). The addition of rFVIIa did not influence platelet deposition but raised fibrin formation and thrombin generation at both shear rates (P < 0.05). At 5000 s(-1), closure times (CT) in the PFA-100 were significantly shortened in the presence of hTF (154.09 +/- 14.69 s vs. 191.45 +/- 16.09 s COL alone; P < 0.05). Addition of rFVIIa did not cause a further reduction of CT. CONCLUSIONS: Our studies demonstrate that hTF is an adhesive substrate for platelets and suggest that the von Willebrand factor could mediate these interactions. At low and intermediate shear rates, rFVIIa enhanced the procoagulant action of hTF, but this effect was not observed at very high shear rates.

In vitro evaluation of platelet reactivity toward annuloplasty devices treated with heparin coating: studies under flow conditions.

We have applied an in vitro perfusion model to explore the potential thrombogenicity of polyester annulolasty fabric used in valve repair and to investigate the possible thromboresistance characteristics conferred by a special heparin coating (Duraflotrade mark treatment). Samples of human blood from i) untreated or ii) heparin-coated extracorporeal circuits were recirculated through annular perfusion chambers containing a) untreated or b) treated annuloplasty cloth material. Perfusion experiments were performed at a shear rate of 600 s(-1) for 20 min. Platelet interaction with the material was morphometrically evaluated. In experiments performed with blood from untreated circuits and cloth material, the average cross-sectional area of platelet mass was 615 +/- 135 microm2. Treatment of cloth material with Duraflotrade mark statistically decreased the area of interacting platelets to 319 +/- 101 microm2 (*p < 0.05, n = 10). Blood samples from heparin-coated extracorporeal circuits showed a decrease of total area of platelets (308 +/- 58 microm2 vs 138 +/- 30 microm2, *p < 0.05, n = 9). The combined treatment of Duraflotrade mark in extracorporeal circuits and cloth material caused a more consistent reduction (p < 0.05). The in vitro perfusion experimental model was sensitive to evaluate the thrombogenic potential of Duraflotrade mark treatment. Our results indicate that the heparin coating of cloth material and extracorporeal circuits improves the biocompatibility of the original material and reduces the thrombogenic profile.

Effects of a new pathogen-reduction technology (Mirasol PRT) on functional aspects of platelet concentrates.

BACKGROUND: Several strategies are being developed to reduce the risk of pathogen transmission associated with platelet (PLT) transfusion. STUDY DESIGN AND METHODS: The impact of a new technology for pathogen reduction based on riboflavin plus illumination (Mirasol PRT, Navigant Biotechnologies, Inc.) at 6.2 and 12.3 J per mL on functional and biochemical characteristics of PLTs was evaluated. PLT concentrates (PCs) obtained by apheresis were treated with Mirasol PRT and stored at 22 degrees C. Modifications in major PLT glycoproteins (GPIbalpha, GPIV, and GPIIb-IIIa), adhesive ligands (von Willebrand factor [VWF], fibrinogen [Fg], and fibronectin), activation antigens (P-selectin and LIMP), and apoptotic markers (annexin V binding and factor [F]Va) were analyzed by flow cytometry. Adhesive and cohesive PLT functions were evaluated with well-established perfusion models. Studies were performed on the preparation day (Day 0) and during PCs storage (Days 3 and 5). RESULTS: Levels of glycoproteins remained stable during storage in PCs treated with 6.2 J per mL pathogen reduction technology (PRT) and similar to those observed in nontreated PCs. When 12.3 J per mL PRT was applied, however, levels of GPIbalpha moderately decreased on Days 3 and 5. VWF, Fg, and FVa were not modified in their expression levels, either by treatment or by storage period. Fibronectin appeared more elevated in all PRT samples. A progressive increase in P-selectin and LIMP expression and in annexin V binding was observed during storage of PRT-treated PCs. Functional studies indicated that 6.2 J per mL Mirasol PRT-treated PLTs preserved adhesive and cohesive functions to levels compatible with those observed in the respective control PCs. CONCLUSION: PLT function was well preserved in PCs treated with 6.2 J per mL Mirasol PRT and stored for 5 days.

Antithrombotic effect of a new nitric oxide donor (LA419) on experimental thrombogenesis.

BACKGROUND: The ability of nitrous compounds to donate nitric oxide (NO), an agent with vasodilating and inhibitory effects on platelet function, has been considered a useful pharmacologic strategy for cardiovascular complications. The purpose of this study was to investigate the effects of a new NO donor, LA419, on platelet interaction in an ex vivo model with human blood circulating through collagen-rich surfaces. MATERIALS AND METHODS: Platelet adhesive and cohesive function were analyzed by morphometric procedures after perfusion techniques. Treated blood was exposed to thrombogenic surfaces and platelet interactions were morphometrically evaluated. RESULTS: All the concentrations studied of LA419 (10 microM, 20 microM and 100 microM) reduced overall platelet interaction with a collagen surface (27.19 +/- 4.72; 25.52 +/- 3.52; and 23.44 +/- 3.01, P < 0.05, respectively, vs. 32.31 +/- 1.61% in the control). The antithrombotic effect was confirmed by results in cross-sectional studies performed in arterial vessels exposed to circulating blood. Values of thrombus and covered surface at 20 microM LA419 were, respectively, 13.67 +/- 4.97% and 19.01 +/- 5.89%; respect to controls 34.80 +/- 5.29% and 37.93 +/- 5.34% (P < 0.05). Moreover, LA419 reduced significantly thrombus area (88.45 +/- 21.97 microm(2); P < 0.05) with respect to controls (168.45 +/- 21.97 microm(2)) and thrombus height, from an average of 10.27 +/- 1.05 microm in nontreated blood to 7.16 +/- 0.6 microm in treated samples (P < 0.05). CONCLUSION: From the present data we can conclude that LA419 possesses a strong antiplatelet action, as demonstrated by its ability to significantly inhibit the interaction of platelet with highly thrombogenic collagen surfaces.

Evaluation of effects of rofecoxib on platelet function in an in vitro model of thrombosis with circulating human blood.

BACKGROUND: Cyclooxygenase (COX)-2-selective non-steroidal anti-inflammatory drugs have been used for anti-inflammatory therapy. However, it has also been described that they may increase risk of cardiovascular events. OBJECTIVES: To study the effects of COX2 inhibitor rofecoxib on platelet function using in vitro tests. Results were compared with those obtained in a parallel experiment with acetyl salicylic acid (ASA). METHODS: Studies of platelet aggregation, using different agonists, were performed by a turbidimetric method. Adhesive and cohesive function of platelets were analyzed by perfusion techniques, treated blood was exposed to thrombogenic surfaces and platelet interaction was morphometrically evaluated. RESULTS: Twenty-five micro M of rofecoxib induced a prolonged lag time and a reduction in the percentage of aggregation when arachidonic acid, ADP or collagen were used as agonists. In perfusion studies with parallel chamber rofecoxib 50 microM and ASA 500 microM reduced overall platelet interaction with the collagen surface (17.4 +/- 3.7, P < 0.05; vs. 32.1 +/- 2.6%P < 0.05 and 17.9 +/- 2.4, vs. 31.9 +/- 3.24, P < 0.05, respectively). In studies performed on annular chambers, 25 micro M of rofecoxib reduced platelet interaction; values of the thrombus and covered surface were 17.4 +/- 4.5%; P < 0.05 and 21.1 +/- 4.1%; P < 0.05, respectively, vs. 30.4 +/- 7.5% and 33.5 +/- 6.5 in the control. ASA did also impair thrombus formation but differences did not reach the levels of statistical significance. Moreover, rofecoxib but not ASA reduced significantly thrombus height and thrombus area (7.4 +/- 0.5 microM; P < 0.005 and 96.0 +/- 21.2 microM(2); P < 0.05 vs. control 11.2 +/- 0.9 microM and 220.0 +/- 47.7 microM(2), respectively). CONCLUSION: We conclude that under our experimental conditions, rofecoxib diminished platelet aggregation induced by different agonists and inhibited platelet-mediated thrombogenesis in an in vitro model of thrombosis.

Recombinant factor VIIa (Novoseven) restores deficient coagulation: experience from an ex vivo model.

The action of recombinant factor VIIa (rFVIIa) in coagulation deficiencies with increased risk of bleeding was investigated using in vitro perfusion. Blood samples were drawn from healthy donors, a patient with hemophilia A and inhibitors, and six patients undergoing oral anticoagulant treatment. Fragmin 10 U/mL was used as anticoagulant. rFVIIa (10 microg/mL in plasma) was added to blood samples, incubated for 1 minute at 37 degrees C, and perfusion studies performed for 10 minutes at 600 x s(-1) through annular chambers containing damaged vascular segments. Subendothelial fibrin and platelets were expressed as a percentage of subendothelial surface screened. Under different conditions, rFVIIa consistently restored or improved fibrin formation on the damaged vascular subendothelium exposed to circulating blood. It restored fibrin deposition in blood from the hemophilia A patient; in patients undergoing acenocoumarol treatment, it reduced the international normalized ratio (INR) from 2.47 to 1.25 with a significant increase in fibrin deposition. Platelet deposition varied slightly between clinical conditions but was less evident in the hemophilia A patient. These data support the concept that rFVIIa facilitates fibrin formation in these clinical situations, promoting procoagulant activity at sites of vascular damage where tissue factor is exposed. This could improve hemostasis in patients with hemophilia A and inhibitors, and in patients treated with oral anticoagulants.

Effect of anticoagulants on activation of polymorphonuclear leukocytes induced by shear stress.

We analyzed how actin polymerization, CD11b expression and homotypic aggregation could be used as markers to study leukocyte activation. Leukocytes were obtained from blood anticoagulated with: citrate, unfractioned heparin (UH) and low molecular weight heparin (LMWH). Flow cytometry was used to study actin polymerization and expression of CD11b after leukocyte exposure to shear stress. Leukocyte aggregation was microscopically assessed. Shear increased both actin polymerization and expression of CD11b in citrated blood (100.1±7.1 vs. 85.8±8.5 p< 0.05 and 53.5±3.5 vs. 20.7±5.1; p< 0.005 respectively). These parameters remained unmodified in UH samples. Using both anticoagulants together, we observed increase in CD11b expression induced by shear stress (59.3±2.1 vs. 25.1±11.0; p< 0,05). LMWH samples showed higher basal levels of actin polymerization and CD11b expression than citrated samples (237±40.8, vs. 85.8±8.5 p< 0.05 and 47.8±2.6, vs. 20.7±5.1; p< 0.005) but no changes induced by shear were observed. When LMWH was used in combination with citrate we observed a decrease in basal activation and significant modifications in CD11b expression induced by shear stress (80.0±4.1 vs. 50.4±2.7). Leukocyte aggregation was modified by UH at basal levels and by LMWH after shear stress. These results indicate that exposure to shear stress results in leukocyte activation. The choice of anticoagulant is a crucial factor in studies of leukocyte function.

Comparison of the effects of human erythrocyte ghosts and intact erythrocytes on platelet interactions with subendothelium in flowing blood.

We investigated whether ghosts behaved similarly to intact erythrocytes to maintain regular primary hemostasis under flow conditions. To this end we performed perfusion experiments with whole blood in which erythrocytes were replaced by pink ghosts, and platelet interaction with the subendothelial surface of a damaged vessel was morphometrically evaluated. The same objective was sought by means of studies with a platelet function analyzer (PFA-100(TM) instrument). Perfusions performed with control blood reconstituted with intact erythrocytes gave rise to 0.4+/-0.2% contact but not spread platelets, 10.8+/-3.4% adhering and spread platelets, 16.3+/-4.6% platelets in thrombi, with 27.5+/-7.4% of the surface covered. Even though the average diameter of the ghosts was smaller than that of intact erythrocytes (5.3 microm vs. 7.7 microm), the values obtained in perfusions performed with ghosts were similar to those of the erythrocyte controls. Studies performed with the PFA-100(TM) analyzer were consistent with those observed in perfusion studies. The viscosity of control blood was compared with that of blood reconstituted with ghosts. At shear rates lower than 450 s(-1), the viscosity of the ghost samples was higher than that of the controls, but the difference progressively decreased as shear rate increased up to 750 s(-1) (3.61+/-0.15 and 3.71+/-0.17 cP, respectively). In conclusion, the results of our study showed that ghosts behaved similarly to intact erythrocytes in maintaining a normal platelet interaction with digested subendothelium, under conditions of moderate shear rate and constant hematocrit (40%). The rheological activity of ghosts, bodies that are metabolically less active, was sufficient for them to satisfactorily act as substitutes for intact erythrocytes in our system.

A novel surfactant nanoemulsion with broad-spectrum sporicidal activity against Bacillus species.

Two nontoxic, antimicrobial nanoemulsions, BCTP and BCTP 401, have been developed. These emulsions are composed of detergents and oils in 80% water. BCTP diluted up to 1:1000 inactivated>90% of Bacillus anthracis spores in 4 h and was also sporicidal against three other Bacillus species. This sporicidal activity is due to disruption of the spore coat after initiation of germination without complete outgrowth. BCTP 401 diluted 1:1000 had greater activity than BCTP against Bacillus spores and had an onset of action of <30 min. Mixing BCTP or BCTP 401 with Bacillus cereus prior to subcutaneous injection in mice reduced the resulting skin lesion by 99%. Wound irrigation with BCTP 1 h after spore inoculation yielded a 98% reduction in skin lesion size, and mortality was reduced 3-fold. These nanoemulsion formulas are stable, easily dispersed, nonirritant, and nontoxic compared with other available sporicidal agents.